Bone Morphogenetic Protein as Bone Additive around Dental Implant and its Impact on Osseointegration: a Systematic Review

Statement of the Problem: Bone morphogenetic protein (BMP), a potential osteoinductive agent, was systematically reviewed for merits and demerits when used as a bone additive that was intervened during the surgical phase of dental implant placement; and suitable drug carriers that could withstand the functional load and deliver BMP at its lowest concentration. Purpose: To identify the carriers and concentration of BMP acceptable during surgical phase of implant placement and evaluate its efficacy in bone gain and osseointegration. Materials and Method: The study design was systematic review. Literature search as per PICO format was carried out within a time range from 2000 to July 2021. The review fol-lowed PRISMA guidelines and registered with the PROSPERO (CRD42020171667). The focus question included the population with an intra-oral implant placed in both animal and human models that were intervened with BMP-2 as an external additive biomaterial during the surgical phase. 2631 articles selected from the initial search were systematically filtered and yielded 16 articles that were qualitatively analysed Results: The inter-rater reliability and level of agreement were 93.71%, κ(Kappa)>0.81 re-spectively. Results revealed the collagen carrier was commonly used for BMP delivery but lacked the property to withstand functional load and sustained release. BMP concentration varied in the range of 0.215μg to 0.8mg and the study revealed significantly indifferent out-come with low dose compared to the highest dose. BMP supplement showed better osseointe-gration in comparison with non-supplemented sites during the early period (within 6 months). Conclusion: BMP at lower concentrations and with appropriate carriers, collagen sponge, hydroxyapatite/tricalcium phosphate (HA/TCP) with a bio ceramic bulking agent, and poly (D, L-lactide-co-glycolic acid) (PLGA) reinforced with gelatin/HA/TCP accelerated bone growth during the initial stages of healing. Further long-term clinical trials for dental implant, analysing the sustained release of BMP with biodegradable and load-bearing carriers should be considered.


Introduction
Long-term survival of implant requires balanced bone remodelling to maintain the bone architecture and osseointegration [1][2]. Bone remodelling occurs by the process of resorption and deposition of bone around the implant, and it begins from the early phase of implant healing [3]. Osteotomy followed by torquing of the implant in the bone initiates osteoclastic differentiation to remove the dead bone and debris by releasing fibroblast growth factor-2, interleukin-1, interleukin-6 and macrophage colony-stimulating factor [4]. The osteoclastic activity is followed by the release of growth factors like insulin-like growth factor, platelet-rich fibrin, bone morphogen-etic protein (BMP) to promote vascularizat-ion and osteogenesis to remodel the peri-implant bone [5].
The imbalance in the release of bone growth promo-ting cells alters bone remodelling and accelerates the bone loss around dental implants. Hence, the addition of bone growth-promoting factors or bone additives will fasten the bone formation by promoting osteogenesis [6][7].
Grafting with growth-promoting factors or bone substitute materials regenerates the bone through either osteo-induction or osteo-conduction [6]. The primary choice of bone substitutes were autogenous grafts as it has the ability of osteo-induction, which is differentiation of immature cells to osteoblast [7]. However, due to limitations in harvesting the autogenous grafts, allografts, xenografts, and alloplasts came into existence [6,8]. Allografts and xenografts have varied host cell acceptance while, the alloplastic graft has only osteo conductive potential (acts as a scaffold) [9]. Hence, alternative materials were searched for the achievement of osteo-induction without host rejection of the graft that promotes bone cell migration, differentiation, and proliferation to enable bone remodelling [10].
BMP are growth factors, belonging to the transforming growth factor-β family, induces new bone formation by inducing differentiation of multi-potent cells [11][12].
BMPs were first identified when demineralized lyophilized bone was incorporated in ectopic site induced bone formation, and in 1971, Urist [13] addressed the role of BMP's in osteo-induction. Further research succeeded in cloning the genes, which code for BMP [14]. BMP forms about 1 part per billion of the bone, and were also isolated from demineralized bone matrix, Escherichia coli, osteosarcoma cell line [13,[15][16]. Though 20 types of BMP were identified, BMP-2, 7, 8 and 9 were proved effective in enhancing osteogenic activity; and BMP 2 and 7 are approved for human use [14,[17][18].
The influence of BMP in wound healing, repair, and new bone formation is most widely researched in the field of orthopaedics [19]. In dentistry, the literature reveals that BMP is widely used as a bone augmentation material or an implant surface modifier [20][21]. BMP was delivered as a bone growth additive during the surgical phase of dental implant placement would accelerate the bone remodelling [22]. BMP per se is efficient in bone regeneration but the use of carriers helps in local delivery and also reduces the concentration of BMP required for its action in the grafted site [23]. Carriers retain the BMP for a longer duration and allow a sustained release to ensure healing and regeneration completion [24]. The carriers are selected based on their biodegradability and osteo-inductivity [23][24]. Though the efficacy of BMP in bone regeneration is evident, its effect around the implant region is unclear. Implant commonly made of titanium has the potential to alter cell infiltration. In addition, bone adjoining the dental implant receives functional load and the carrier utilized should have sufficient strength to withstand the force [25]. Hence, a literature search was carried to identify an appropriate carrier and concentration of BMP that would be effective around the dental implant for bone regeneration during surgical placement. The review involved both animal and human models to identify the newer trends developed in animals, which is still a void in a human clinical trial. This systematic review exclusively evaluated the types of carriers and concentration of BMP in promoting osseointegration when used as a bone growth additive during the surgical phase of implant treatment.

Materials and Method
The systematic review was conducted in accordance

Inclusion criteria
The eligibility for inclusion of the articles in the review was considered only when the BMP was used as a bone additive along with surgical placement of an implant in the oral cavity. Only prospective studies involving animal and human models with a minimum follow-up period of six months were included. Implant placement in any other region was not considered in the systematic review. In addition, the review did not include data when BMP was used without placement of the implant or as an implant surface modifier. Experimental comparative study design with intervention for animal experiments was included in the review. To evaluate the highest level of evidence in human research, randomized controlled trials were included. This was conducted to analyse the deficiency in human research and the scope of future research in a clinical trial.

Data collection
The data were extracted by the first author (FB) and filled into a pre-defined form that evaluated the basic characteristics of the study: authors, year of publish, study design, aim and outcome. The data extracted were tabulated chronologically and the data synthesis was based on evidence tables and descriptive summaries ( Table 1, 2). The second author (SK) checked the information collected, and the third author (AK) settled the disagreement between the authors.

Risk of bias assessment
The "systematic review centre for laboratory animal experimentation (SYRCLE) RoB tool" updated March 2014 assessed the risk of bias for animal studies ( Table   3). The bias assessment for randomized controlled trials was performed using "revised Cochrane risk of bias tool for randomized trials (RoB2)" version of 22 August 2019 (Table 4).

Summary measures and data synthesis
The data were summarised based on the type of samples (animal or human), the number of samples and implants, comparisons, follow-up months, type of carrier, the concentration of BMP, the methods of investigation, and outcome measured ( Table 2). The outcome of the hypothesis; osseointegration around the dental implant was summarised qualitatively based on the type of bone, mineralization, bone density, bone height, bone-implant contact, and implant stability. Since there was no common outcome or measurement between the collected articles, quantitative measurement was not performed.
The inter-rater reliability and kappa statistics were performed for agreement between the authors for the eligibility and inclusion section. The inter-rater reliability for FB and SK, SK and AK, AK and FB were 92.64%, 94.34%, and 94.34% respectively. The inter-rater reliability between the three authors was 93.71%. The kappa analysis of agreement between FB and SK, SK and AK, AK and FB were 0.81, 0.84, and 0.84 respectively suggesting almost perfect agreement.

Study selection
The search yielded 2631 articles (includes 419 Pubmed, 2137 Science Direct, 12 Cochrane, 56 Embase indexed articles, and also included 7 Opengray literature). 735 duplicate articles were filtered out and the remaining   The data extracted were tabulated in Table 2.

Carriers and Concentration of BMP
In the animal model, the delivery of rhBMP-2 around dental implants varied from calcium phosphate deriva-    [32]. The lowest concentration of 0.2 µg was tried in an animal study with a calcium phosphate carrier [38]. In human studies, the BMP carriers used were xenogeneic bone grafts, collagen and HA/ TCP with 0.5mg/ml, 1.5mg/ml, 0.25mg/ml of BMP respectively [22,[39][40].

Measuring outcome(osseointegration) based on concentration and carriers
The evidence of the efficiency of carriers and varied co- had values of 8 and 11.5 without the use of carriers [28], whereas an increase of concentration of 20µg and 50µg with HA/βTCP/collagen carrier had establis-hed a value of more than 75 [34][35]. Use of concentration less than 20 µg by Smeets et al. [38], (0.3µg), Huh et al. [28], (10, 20µg) and Chao et al. [34], (5,20, 50µg) did not show significant improvement in osteoconductive potential compared to the control site, wherein the control site was the carrier. The concentration of BMP used in the reviewed articles varied between the range 0.215µg to 0.8mg and the outcome of the studies showed improved bone formation around implants despite the concentration, however, a significant effect was observed when concentration was above 20µg.
The bone density for the coronal and soak-loaded implants were 38 % and 34% respectively without the use of carriers [28], whereas the density of the peri-  [36].

BMP-2
The animal experiments revealed the efficiency of the BMP group compared with a non-BMP group. Chang et al. [37] revealed the absence of osteogenesis and reduced mineralization in the non-BMP group at 4 weeks.
Lyu et al. [31], had also observed an insignificant increase in bone gain at 4 weeks, while Schorn et al. [32] and Chao et al. [34], also observed the same; they revealed maximum gain with BMP was achieved at 12 wee- between BMP and non-BMP groups respectively [32]. Whereas with HA/βTCP/Collagen increase in bone vol-ume density was insignificant, that varied between 50-60% with the highest percent for the BMP group [34].
Similar trends were also seen in human studies [22,39]. Jung et al. [22] stated that the new bone formation with rhBMP-2 using xenogeneic bone substitute carrier was a mixed type including randomly arranged fibres of woven bone and the parallel fibre orientation of lamellar bone. This comparative analysis between the xenogeneic bone substitute with rhBMP-2 (test) and without rhBMP-2 (control) revealed that the average bone density was 37% and 30% respectively and mineralized bone (lamellar) were 76% and 56%, respectively in the periimplant region [22]. between the non-rhBMP-2 and rhBMP-2 groups was observed at 6 months, became statistically insignificant at 5 years, with only a mean change of 0.2mm [22,36].
The carriers and concentration in each of the studies differed and hence meta-analysis was not conducted in this systematic review for quantitative synthesis of the result.

Discussion
BMP-2, an osteo-inductive protein, has the ability to improve bone formation with a property similar to autogenous bone grafting [41][42]. For effective cellular ingrowth and stabilization at the grafted site, the osteoinductive proteins require a delivery system or a carrier [24]. The combination of an osteoconductive carrier such as xenogeneic bone substitute with an osteoinductive protein was effective in improving bone regeneration [22,39]. However, xenogenic bone substitute are antigenically dissimilar to human cells and can induce an immunogenic response [43]. The biomaterials such as collagen sponges, hydroxyapatite, fibrin, alginate, hyaluronic acid, and synthetic polymers were tried in both orthopaedic and maxillofacial bone augmentation [44][45][46][47]. Though collagen is neither osteoconductive nor can withstand the functional load, it is considered one of the best-described carrier materials for the growth factor [48].
On critical evaluation between the studies, collagen membrane was frequently used as a carrier for BMP.
The review revealed that the collagen membrane as a carrier enhanced the osteo-inductivity of BMP-2 in the peri-implant region because of its ability to confine the growth factor in close proximity with the periosteum [22,32,36,39]. Fixation of collagen membrane with pins prevented mechanical failure, improved its stabilization and the efficiency of BMP-2 [36]. The use of carriers namely HA/TCP with bio ceramic bulking agent withstood the functional load and promoted the bone regeneration comparable to collagen sponge [25,37]. HA/TCP used in varying ratios modified as bicalcium phosphate was proved to improve the bioactivity and mimics as the natural bone without immunogenic response [33]. HA/βTCP without BMP and with BMP did not show a significant difference in their bioactivity until 4 weeks in the majority of the research, whereas effective change was observed after 8 weeks [32][33][34][35].
Cottam et al. [27], stated that the carriers were apparent-  [26,28]. In addition, the researchers who worked independently with much lesser concentration at microgram level (.215µg) also proved the effectiveness of BMP in bone regeneration with the use of carrier HA/βTCP in PLGA [37]. In contrast researchers with calcium phosphate as a carrier did not find a significant difference at a lower concentration of 2µg of BMP [38]. Chao et al. [34], claimed that between 2, 20 and 50µg in HA/βTCP/ Collagen, 50µg of BMP-2 significantly increased the osseointegration.
Wikesjö et al. [50] reported that a high concentration created a radiolucent area around an implant that did not affect the bone formation. They also stated that the immature bone was abundant with higher concentration while the mature bone was observed with lower concentration [50]. Though rhBMP-2 accelerated the minerali-